Acrosomal sperm protein and uses thereof

ABSTRACT

The present invention relates to the use of acrosomal sperm protein in immunocontraception of male and female subjects and uses thereof as a marker for fertility.

BACKGROUND OF THE INVENTION

(a) Field of the Invention

The invention relates to the use of acrosomal sperm protein inimmunocontraception of male and female subjects and uses thereof as amarker for fertility.

(b) Description of Prior Art

Fertilization is a highly orchestrated process that culminates in theactivation of an oocyte by a spermatozoon. Although testicularspermatozoa are fully differentiated cells, they cannot efficientlyencounter the oocyte's investments. In order to acquire this property,spermatozoa must undergo post-testicular modifications within theepididymis. During this transit, the male gamete is subjected to majorsurface modifications such as changes in the lipids composition,acquisition of new epididymal proteins as well as post-translationalmodifications of sperm proteins. Taken together, these modifications areprerequisites for the spermatozoon to acquire its fertilizing ability.These processes are regulated by the epididymal luminal microenvironmentwhich is influenced by both epididymal and testicular protein synthesisand secretion.

Using the hamster as a model, we have previously described a 26 kDaprotein, the P26h, which shows immunocontraceptive properties when usedto actively immunize male hamsters (Berube, B., Sullivan, R., 1994,Biol. Reprod., 51: 1255-1263). This protein is localized on the spermacrosome and is acquired during the epididymal transit. P26h plays arole in egg sperm interactions as shown by the ability of P26hantibodies to inhibit sperm-zona pellucida binding in vivo and in vitro(Berube, B., Sullivan, R., 1994, Biol. Reprod., 51: 1255-1263).

It would be highly desirable to be provided with to the use of anacrosomal sperm protein in immunocontraception of male and femalesubjects.

It would be highly desirable to be provided with to the use of anacrosomal sperm protein as a marker for fertility.

SUMMARY OF THE INVENTION

One aim of the present invention is to provide the use of acrosomalsperm protein in immunocontraception of male and female subjects.

In accordance with the present invention there is provided a method ofimmunocontraception of a male or female subject, which comprisesadministering to said male or female subject an antigenic amount of P34or an antigenic fragment thereof to elicit an immunocontraceptionresponse by said male or female subject.

The preferred P34 protein used has the sequence identified as SEQ IDNO:3 and the preferred antigenic fragment thereof includes, withoutlimitation, MELFLAGRRVC (SEQ ID NO:4) OR CSQDYAEPNPTWQV (SEQ ID NO:5).

An immunocontraceptive vaccine for male or a female subject, whichcomprises an antigenic amount of P34 or an antigenic fragment thereof inassociation with a suitable pharmaceutically acceptable carrier, whereinsaid vaccine elicits an immunocontraception response by said male orfemale subject after its administration.

In accordance with the present invention there is provided a probe as amarker for male or female fertility, which comprises a cDNA sequencecapable of hybridizing under stringent conditions with human acrosomalsperm protein P34.

In accordance with the present invention there is provided a method forthe diagnosis of male or female infertility which comprises the stepsof:

a) determining the amount of human P34 in a sperm or ovule sample; and

b) comparing the determined amount of step a) with a fertile controlsample.

The amount of human P34 in step a) may be determined using an antibodyraised against human P34.

In accordance with the present invention there is provided a kit for thediagnosis of male or female infertility which comprises:

a) an anti-P34 antibody enzyme-labeled;

b) an enzyme substrate; and

c) a fertile control sample.

A calibration curve for the amount of human P34 may be obtained usingthe fertile control sample of component (c) above.

For the purpose of the present invention the following terms are definedbelow.

The term "antigenic fragment" is intended to mean any fragment of saidprotein which is capable of eliciting an immune response pursuant to itsadministration to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the comparison of partial amino acid sequences withthe corresponding amino acid sequence of P26h with AP27;

FIG. 2 illustrates a Northern blot analysis of hamster total RNA from 1)testis 2) whole epididymis 3) caput epididymis 4) corpus epididymis 5)cauda epididymis 6) Fat 7) Lung 8) heart 9) liver 10) kidney 11) muscle12) brain probed with a nP26h 710 bp cDNA probe (upper panel) or with apositive Cyclophilin probe (lower panel);

FIGS. 3A-3B illustrate the nucleotide sequence of the P26h cDNA;

FIG. 4 illustrates the alignment of the deduced amino acid sequence ofP26h with the AP27 and the Carbonyl Reductase;

FIG. 5 illustrates in situ hybridization probed with the P26h RNAsprobes;

FIG. 6 illustrates the immunoprecipitation of P26h cDNA translationalproducts;

FIG. 7 illustrates a Northern blot analysis of human total RNA from 1)testis, 2) caput epididymidis, 4) corpus epididymidis, 5) caudaepididymidis, probed with a P34H cDNA probe; and

FIG. 8 illustrates the sequence homology of the human P34 (lower lane)counterpart of P26h (upper lane).

DETAILED DESCRIPTION OF THE INVENTION

During fertilization, mammalian spermatozoa must undergo a series ofevents in order to reach the oocyte surface and to perform syngamy. Someof these events occur during the epididymal transit where spermatozoaacquire their fertilizing ability. We have previously described ahamster sperm protein, P26h, acquired during the epididymal transit.P26h shows immunocontraceptive properties when used to actively immunizemale.

In accordance with the present invention, we have undertaken thedetermination of the origin and of the sequencing of the encoding cDNAof this sperm protein showing male contraceptive properties. N-terminalsequencing of purified P26h and of peptides generated by partialproteolysis allowed partial identification of the protein. Northern blotanalysis revealed that a major transcript encoding for P26h waslocalized the testicular MRNA whereas no signal was detectable in othersomatic tissues of the hamster. A hamster testis cDNA library wasscreened and a P26h encoding cDNA was cloned and sequenced. The P26hcDNA sequence revealed a 85% identity with the cDNA corresponding tomouse Adipsin and of a Carbonyl Reductase. The deduced P26h amino acidsequence possesses specific domains of the Short ChainDehydrogenase/Reductase (SDR) family proteins. Antibodies generatedagainst synthetic peptides deduced from the cDNA sequence recognized theP26h on Western blots of detergent extracted hamster sperm proteins. Onthe other hand, in vitro translational products synthesized from theP26h cDNA were immunoprecipitated by a polyclonal antiserum producedagainst the purified hamster sperm P26h. In situ hybridization performedon tissues from the hamster reproductive tract, revealed that the P26hwas principally transcribed in the seminiferous tubules and at a lowerlevel in the corpus epididymidis. P26h shows unique features of the SDRfamily that can be used to induce contraception in males.

Materials and methods

Animals

Sexually mature Golden hamsters (Mesocricetus auratus; Charles RiverInc., St. Constant, Qc, Canada) were used in this study. Hamsters weresacrificed under CO₂ atmosphere, the epididymidis were excised, defattedand dissected into caput, corpus and cauda segments. Tissues were frozenin liquid nitrogen and stored at -80° C. until use. Testicular andsomatic tissues were proceeded the same way. For in situ hybridizationfresh tissues were rinsed in PBS-DEPC (Phosphate buffered saline-Diethylpyrocarbonate) and fixed at 4° C. for 2 h in 4% (w/v) paraformaldehydefreshly prepared in PBS. Tissues were cryoprotected by sequentialincubations in 10% glycerol for 1 h at 4° C. under agitation and thenovernight in 50% OCT. Tissues were embedded in OCT and frozen in liquidnitrogen. Cryosections of .sup.˜ 7 μm were collected on poly-L-Lysinecoated slides, air-dried at -20° C., and stored at -80° C. until used.

N-chlorosuccinimide proteolysis

Proteins from cauda epididymal spermatozoa or from the epididymal fatpad were extracted with 0.5% Nonidet™ P40 (Sigma) as previouslydescribed and submitted to preparative SDS-PAGE. After Coomassie bluestaining, the bands corresponding to a MW of 26 kDA were excised, washedtwice with H₂ O, and rinsed with a washing solution (50% (wt/vol) urea,50% (vol/vol) ethanol). The polyacrylamide bands were incubated 30 min.in 20 mg/ml N-Chlorosuccinimide in washing solution, washed in water,and then incubated 3 times for 1 hour each, in an equilibrium solution(0.0625 M Tris-HCl pH 6.8, 20% (vol/vol) glycerol, 30% (vol/vol)B-mercaptoethanol, 6% (wt/vol) SDS). The band was loaded on adiscontinuous polyacrylamide gel and submitted to electrophoresis.Patterns of proteins fragments were visualized by silver nitratestaining, or Western blotted using a P26h antiserum (Berube, B.,Sullivan, R., 1994, Biol. Reprod., 51: 1255-1263). Western blotted P26hfragments were also used for N-terminal sequencing as described below.

Partial amino acid sequence analysis

P26h was purified and absorbed on a piece of nitrocellulose sheet. Onehundred μl of 50 mg/ml CNBr (Cyanogen Bromide) in 70% formic acid wasadded to 1 mg of the dry protein and incubated under nitrogen in thedark for 24 h. Digested peptides were loaded onto a VYDAK™reversed-phase C18 column (250×1 mm) which was equilibrated with 0.1%(v/v) trifluoroacetic acid (TFA) in water and eluted with a 2-100%gradient of 0.08% (v/v) TFA in 80% acetonitrile. Fractions of 0.5 ml orsmaller were collected at a flow rate of 50 μl/min. Protein sequence wasperformed on aliquots from one peak by automated Edman™ degradation witha pulsed-liquid phase sequencer.

RNA extraction

Tissues were homogenized with a Polytron™ in 1.5 ml of a freshhomogenization buffer solution (4 M guanidium thyocyanate, 25 mM sodiumcitrate pH 7, 0.5% sarcosyl, 0.1 M 2-mercaptoethanol). One ml of CesiumChloride-homogenization buffer (2 g of CsCl/2,5 ml) was added to thetissue lysates. This was layered on cushion solution (5.7 M CsCl, 0.1 MEDTA, pH 7.5) and centrifuged at 60,000 g overnight. The RNA pellet wasresuspended in TES solution (10 mM tris-HCl, 5 mM EDTA, 1% SDS, pH 7.4)and extracted with phenol/chloroform and chloroform/alcohol isoamyl24:1. RNA was precipitated with 0.1 vol. of sodium acetate (3 M, pH 5.2)and 2.5 vol. of ethanol 95%. The RNA pellet were resuspended in DEPCwater. The RNA quality was evaluated by electrophoresis on a 1% agarosegel. All solutions were made with DEPC water.

Northern blot analysis

Total RNA (20 μg) prepared from hamster and human tissues wereelectrophorized on 1% agarose-formaldehyde gels and transferred to anylon membrane (Quiagen, Santa Clarita, Calif.) using 20× SSC (3 M NaCl,0.3 M Na-Citrate). Air dried Northern blots were UV cross-linked andprehybridized at 42° C. for 4 h in 50% (vol/vol) formamide, 0.75 M NaCl,0.05 M NaH₂ PO₄, 0.005 M EDTA, 2× Denhardt's reagent [0.2% (wt/vol)Ficol 400, 0.2% (wt/vol) polyvinylpyrrolidone, 0.2% (wt/vol) BSA], 0.2mg/ml herring sperm DNA (Sigma Chemicals, Mississauga, ON) and 0.1% SDS.The membrane was hybridized overnight at 42° C. in the same solution, towhich [α-³² P] dCTP-labeled DNA probes were added. The membranes werethen washed twice in 0.1× SSC-0.1% SDS followed by a third wash of 30min. at 65° C. in 0.1× SSC-0.1% SDS, and exposed on Kodak™ X-O-Mat filmwith intensifying screens for 6-18 h at -80° C. A RNA ladder (1.6-7.4kb; Boehringer Mannheim, Laval, QC) was electrophoresed in parallel andCyclophilin probe was used as a constitutive internal control.

RT-PCRs production of a P26h cDNA probe

The first amino acids sequence obtained (MKLNFSXLRLVTGAGKGIG) showedhigh homology with the peptide sequence of the Adipsin: a marker ofadipocytes differentiation. From the nucleic acid sequence of theadipsin, two primers were selected according to OLIGO 4.01™ primeranalysis software (National Biosciences, Plymouth, Minn.), chemicallysynthesized (sense downstream 5'-GTG ACA GGG GCA GGG AAA GGG-3' andantisense upstream 5'-GCA ACT GAG CAG ACT AGG AGG-3') and used forRT-PCR on the total RNA from hamster's testis.

Briefly, 5 μg of the total testis RNA were incubated with 0.5 μg oligodeoxythymidine primer at 70° C. for 10 min. in a final volume of 12 μland then kept on ice. Samples were then incubated for 60 min. at 42° C.in a reaction mixture containing 4 μL of 5× buffer (250 mM Tris-HCl, 375mM KCl, 15 mM MgCl₂), 10 mM dithiothreitol (DDT), 1.25 mMdeoxynucleotide triphosphates (DNTP) and 200 U Super Script reversetranscriptase in a final volume of 20 μl. Expression of the P26h genewas determined by amplification of the cDNA. Each reaction contained 5μl of RT template (or water as negative control), 1.5 mM MgCl₂, 1×buffer, 0.2 mM dNTPs, 10 μM of each primer and 1-5 U Taq polymerase(Pharmacia Biotech, Baie D'Urfe, QC) in a final volume of 50 μl. The PCRcycling conditions chosen were 1 min. at 95° C., 1 min. at 60° C., 1min. at 72° C. for 30 cycles, followed by a 5 min. extension at 72° C.The reaction products were analyzed using electrophoresis on a 1%agarose gel; the bands were visualized by ethidium bromide staining.

The PCR band (.sup.˜ 710 pb) was purified (Quiaquick; Quiagen), T-Clonedin pCR 3.5 (Invitrogen, San Diego, Calif.), and digested with EcoR1. Theinsert (710 bp) was separated from the vector and other fragments byelectrophoresis on a 1% agarose gel, isolated from gel matrix with Na45membrane (Schleicher & Schuell, Inc.), and random-prime labeledaccording to the supplier's instruction using the T7 Quick-Prime™ kit(Pharmacia Biotech, Baie D'Urfe, QC) with [α-³² P] dCTP. CyclophilincDNA was also random-prime labeled using the same procedure.

Cloning and sequencing of P26h cDNA

Poly(A)⁺ RNA from hamster and human testicular tissues was purified fromtotal RNA solution using a poly(A)⁺ RNA purification kit (PharmaciaBiotech, Baie D'Urfe, QC) according to the supplier's instructions. ThecDNA library was prepared according to the instruction the of thesupplier. Briefly, testicular poly(A)⁺ RNA was reverse-transcribed andligated into the lambda Uni-Zap XR vector (Stratagene, La Jolla,Calif.). The lambda library was packaged and amplified using Escherichiacoli XL1-Blue cells, and screened with the random-prime labeled 710 bpP26h cDNA. The positive clones were isolated by plaque purification andthe longest one (1081 bp) was subcloned into pBluscript KS+. Allnucleotide sequences were determined by the dideoxinucleotidetermination method (Sanger) using T7 Sequenase v 2.0 kit. The labeledreaction products were analyzed on a DNA sequencer gel. Sequencetranslation was performed using Gene Jockey software (Biosoft,Cambridge, UK).

In situ hybridization

Tissues cryosections were fixed with freshly prepared 4%paraformaldehyde in PBS for 5 min at RT° (Room Temperature), incubatedfor 10 min. in 95% ethanol/5% acetic acid at -20° C., and rehydrated bysuccessive baths of decreasing concentrations of ethanol diluted withDEPC-H₂ O. Target RNA was unmasked by enzymatic digestion with 10 μg/mlproteinase K (Boehringer Mannheim) in PBS for 10 min. at 37° C.,followed by a 5 min. incubation in 0.2% glycine. Sections were postfixedfor 5 min with 4% paraformaldehyde in PBS, acetylated with 0.25% aceticanhydride, 0.1 M triethanolamine, pH 8.0, for 10 min., and finallywashed with PBS.

Tissues were prehybridized for 1 h with a preheated 250 μg/ml salmonsperm DNA in a hybridization solution (0.3M NaCl, 0.01 M Tris-HCl pH7.5, 1 mM EDTA, 1× Denhardt's solution, 5% dextran sulfate, 0.02% SDSand 50% formamide). Sections were then incubated overnight at 42° C.,under coverslips, with 25 μl of heat-denatured antisense or sense CRNAprobed with DIG (Digoxigenin: Boehringer Mannheim) according tosupplier's instruction. Coverslips were removed, the sections werewashed twice in 2× SSC at RT°, followed by two 10 min. washes at 42° C.in 2× SSC, 1× SSC and 0.2× SSC, respectively.

Hybridization reactions were detected by immunostaining with an alkalinephosphatase-conjugated anti-DIG antibobies. Nonspecific staining wasblocked by incubation for 1 h with 5% (v/v) heat-inactivated sheep serumin 0.2 M Tris-HCl, 0.2 M NaCl, and 3% Tritons X-100. Sections were thenincubated for 2 h at RT° with the alkaline phosphatase-conjugatedanti-DIG antibodies diluted 1:1000 in blocking solution, washed withtris-HCl/NaCl buffer, and incubated with 0.1 M tris-HCl, pH 9.5, 0.1 MNaCl, and 0.01 M MgCl₂. The hybridization signal was visualized after a10-15 min. incubation with the substrates nitroblue tetrazolium chlorideand 5-bromo-4-chloro-3-indolylphosphate p-toluidine salt (GIBCO-BRL,Gaithersburg, Md.). Levamisole (2 mM; Sigma) was added to the reactionmixture to inhibit endogenous alkaline phosphatase. Slides were immersedin 1 mM EDTA, 0.01 Tris-HCl, pH 7.5, washed 5 min. in H₂ O,counterstained with neutral red, dehydrated through baths of ethanol,cleared in xylene, and mounted with Permount (Fisher scientific, Nepean,Ontario, Canada).

Eukaryotic in vitro translation

In vitro translation was performed from circular plasmid DNA includingthe P26h cDNA. The TNT coupled reticulocyte lysate system was usedaccording to the supplier's instructions (Promega, Madison, Wis., USA).Briefly, 0.5 μg of circular plasmid DNA was added directly to TNT rabbitreticulocyte lysate. T₃ RNA polymerase (Promega, Madison, Wis., USA) andS³⁵ -methionine (10 mCi/ml) were added to the translation mixture. Thereaction was performed for 2 hours at 30° C. The de novo synthesizedproteins were analyzed by SDS-PAGE according to Laemmli. The gel wassoaked in an enhancer solution (Amersham), dried, and exposed on X-Omat™AR film (Kodak) for 6 hours at room temperature. In some experiments,the translational products were submitted to NCS proteolysis (asdescribed above) before electrophoretic analysis.

In some experiments, the translational products were immunoprecipitatedusing an anti-P26h antiserum. 5 μl of the translation reaction mixtureswere incubated 1 h at room temperature with the P26h antiserum(previously described Berube, B., Sullivan, R., 1994, Biol. Reprod., 51:1255-1263) or the control serum, both diluted in Tris-saline (50 mMTris-HCl, 150 mM NaCl, pH 7.5). 50 μl of packed protein-A sepharose(Pharmacia) was added for 1 hour at room temperature. Theimmunoprecipitate was washed several times in tris-saline solution (50mM Tris-HCl, 500 mM NaCl, pH 7.5). The immune complexes were dissociatedin SDS-PAGE sample buffer (50 mM Tris-HCl pH 6.3, 2% SDS (w/v) and 5%(v/v) B-mercaptoethanol) and submitted to SDS-PAGE according to Laemmli.The gel was enhanced, dried, and exposed on X-Omat™ AR film (Kodak) for12 hours at room temperature.

Results

When purified P26h was submitted to Edman degradation, 27 of the 29amino acids generated were identified. A 17 kDa fragment obtained by NCSproteolysis of P26h allowed the identification of 15 of the 26 aminoacids analyzed, whereas the fragment obtained following CNBr treatmentallowed the identification of 8 of 9 additional amino acids. For a totalof 40 amino acids identified by Edman degradation of P26h peptides, 37showed homology with a mouse Adipsin sequence (FIG. 1). This protein hasbeen shown to be a differentiation growth factor of mouse adipoblasts.

In the hamster, like many mammalian species, the epididymis issurrounded by a fat pad. In the mouse, an Adipsin mRNA is abundant inthe epididymal fat cushion. We were concerned with the possibility thatP26h N-terminal sequences obtained may result from a contamination ofsperm protein preparation by epididymal fat pad originating Adipsin. Aband corresponding to 26 kDa of an electrophoretic pattern of proteinextracted from a large amount of epididymal fat cushion was excised andsubmitted to N-chlorosuccisinimide proteolysis (NCS). This digestion didnot generate fragments on SDS-PAGE electrophoretic pattern whereas P26hNCS digest generated a 22.4 kDa fragment. Moreover, only the P26h andits NCS digested fragment were detected by a P26h antiserum used toprobe a corresponding immunoblot. The NCS digested fragment of the P26hwas sequenced and the inner sequence revealed also a high homology levelwith the Adipsin. The intact 26 kDa protein from the fat pad proteinextract was submitted to the same procedure and no N-terminal sequencewas obtained.

RT-PCRs were performed with oligonucleotides derived from the cDNAsequence of the Adipsin. A 710 bp fragment was amplified from thehamster testis, cloned and sequenced. The sequence revealed that thisfragment has 85% homology with the Adipsin cDNA. Using this fragment asa probe, we performed a Northern blot analysis to determine in whichtissues P26h transcription occurs. Total RNA of several tissues wereextracted and submitted to blot-hybridization analysis The Northernblots showed that the total P26h messenger RNA has 1081 bp and that itwas transcribed exclusively in the testis. To confirm the presence ofintact RNA in all samples, the same blot was probed with randomcyclophilin DNA, and an intense signal was obtained in all samples (FIG.2). By opposition, Northern blot analysis of mRNA prepared from humantissues revealed an abundant transcript in the epididymal tissues (FIG.7).

A cDNA library was constructed in Lambda gtll from the hamstertesticular MRNA and from the human epididymal tissues. 10⁵ clones of theprimary library (5×10⁵ clones) were directly screened with the 710 pbcDNA probes. The first screening allowed the detection of 32 positiveclones from which 11 were used for a second and third screening. Thesize of the inserts was determined by PCR and the longest insert (clone2), was introduced in pBluescript SK(±) phagemid and sequenced. The P26hcDNA of 1081 bp has a 732 bp open reading frame, starting with a ATGcodon at position 132 and a TAG stop codon at position 764, followed bya poly-adenylation signal, and a poly A tail (FIGS. 3A-3B). The sequenceis numbered from the 5' end of the cDNA clone. The translation of theproposed open reading frame is shown below the nucleotide sequence andencodes a peptide of 244 amino acids terminating by a amber codon.

The deduced amino acid sequence predicted a 26 kDa MW protein which isin agreement with the molecular weight of the P26h as determined bySDS-PAGE. The N-terminal sequence of P26h and of its generated peptidesdetermined by Edman degradation (FIG. 1) were also in agreement with theamino acid sequence deduced from the cDNA (FIG. 4). The P26h amino acidsequence was compared with the Adipsin and a carbonyl reductase, whichshowed a homology of 85% and 86% respectively. Adipsin and carbonylreductase are members of the SDR (short side chaindehydrogenase/reductase) family proteins. The P26h also showed theconserved patterns of SDR, i.e. the NADH or NADPH coenzyme binding siteand the active site which are respectively GlyXXXGlyXGly and TyrXXXLys(FIG. 4). The deduced amino acid sequence of the human homolog (FIG. 8,lower sequence) predicts a 244 amino acid peptide sharing the SDRcharacteristic with the hamster P26h (FIG. 8, upper sequence).

Expression of P26h mRNA is detected in the testis, using non-radioactivein situ hybridization. Digoxigenin-labeled anti-sense probe revealed theexpression of the P26h MRNA in the adult hamster testicular seminiferoustubules. By opposition to the Northern Blot analysis, in situhybridization revealed a weaker signal along the epididymis, principallyin the corpus portion. Digoxigenin-labeled sense probe was used as acontrol for nonspecific hybridization.

Using the TNT™ coupled reticulocyte lysate system, we performed in vitrotranslation with circular plasmid including P26h cDNA. We detected a 26kDa signal with total translation products on SDS-PAGE. (FIG. 6A3) Totaltranslation products were then submitted to immunoprecipitation withanti-P26h antibody, which permitted the detection of a unique signal of26 kDa on SDS-PAGE. (FIG. 6A2). Total translation products were furthersubmitted to NCS proteolysis. The NCS proteolysis generates a 17 kDafragment on SDS-PAGE in agreement with the deduced amino acids sequenceand the previous NCS proteolysis of purified P26h.

Discussion:

During the epididymal transit, the mammalian spermatozoa acquire theirfertilizing ability. One of the best documented physiological functionsacquired by the spermatozoa during epididymal maturation is theirability to efficiently interact with the egg's zona pellucida. Ourlaboratory has been interested by these sperm surface modifications;mainly the addition of new surface proteins, or the post-translationalmodifications of preexisting sperm components, that are necessary toproduce a functional male gamete. Using the hamster as a model, we havepreviously identified a sperm protein, P26h, which shows affinity forthe homologous zona pellucida glycoproteins. P26h is abundant in theluminal fluid of the proximal region of the hamster epididymis, itsconcentration decreasing along the transit. Contemporarily, P26haccumulates on the spermatozoa during the epididymal maturation. P26h isexclusively located on the sperm surface covering the acrosomal cap ofthe mature spermatozoa; the subcellular domain involved in zonapellucida binding.

In accordance with the present invention, P26h has been purifiedfollowing detergent extraction of cauda epididymal spermatozoa. This hasbeen performed by preparative SDS-PAGE (Berube, B., Sullivan, R., 1994,Biol. Reprod., 51: 1255-1263) as well as by chromatographic procedures.In the latter case, a single spot in two dimensional gel electrophoresiswas obtained, this single protein being recognized by the anti-P26h oncorresponding Western blot. These two preparations of purified P26h, aswell as proteolytic fragments, have been N-terminal sequenced by Edmandegradation. All the amino acid sequences obtained showed high homologywith mouse Adipsin (FIG. 1). Adipsin has been described as adifferentiation factor of adipoblasts in adipocytes. Adipsin mRNA hasbeen shown to be present in high quantities in the mouse epididymal fatpad. In the hamster, as well as in the mouse, the majority of theepididymis is surrounded by a fat cushion. Considering that the purifiedP26h was obtained from spermatozoa recovered from the distal caudaepididymidis, we were concerned with the possibility that the N-terminalsequences were obtained from Adipsin liberated from adipocytescontaminating the sperm suspensions. This was conceivable if weconsidered that the Adipsin MW deduced from the mRNA sequence is of 27kDa. Proteins from huge amounts of epididymal fat pad were extracted andproceeded in parallel with cauda epididymal spermatozoa. Protein bandsof 26-27 kDa were excised from preparative SDS-PAGE of proteinsextracted from fat pad adipocytes and from cauda epididymal spermatozoa.Intact 26-27 kDa bands and proteolytic fragments generated by NCS(N-Chlorosuccimide) digest were Western blotted and probed with theanti-P26h serum. The 26-27 kDa fat pad protein was undetectable by theanti-P26h antiserum (FIG. 2). Furthermore, the 26-27 kDa fat pad bandand the P26h sperm protein were proceeded in parallel for N-terminalsequencing by Edman degradation. No signal was detectable when the fatpad protein was proceeded. From these results we can conclude that theN-terminal sequences obtained did not result from a contamination ofsperm preparation by the epididymal fat pad.

Northern blot analysis revealed a major P26h transcript in testiculartissues of the sexually mature hamster (FIGS. 3A-3B). This mRNA isundetectable in the other tisssues analyzed, including the fat pad andthe epididymis (FIGS. 3A-3B). This was unexpected since it waspreviously reported that an in vitro translational product encoded bymRNA of the proximal region of the epididymis can be immunoprecipitatedby anti-P26h antibodies. In this study, in situ hybridization confirmsthat a P26h transcript is predominant in the testis and, at a lowerlevel, in the epididymis. In situ hybridization has been performed withdigoxigenin-labelled RNA probes system using an anti-digoxigeninantibody that allows amplification of the signal and provides a moresensitive mRNA detection than the traditional Northern blot analysis. Afaint labelling is detectable all along the epididymis, a much strongersignal being associated with the corpus (FIG. 5). In many species, thecorpus region is known to be a more active epididymal segment forprotein synthesis and secretion. According to the Northern blot analysis(FIG. 2), P26h which is found at high concentration in the proximalregion of the hamster epididymis probably originates from the testicularfluid as a secretion product of the seminiferous tubules, as suggestedby the in situ localization of the transcript (FIG. 6). This protein mayalso be secreted by the corpus epididymidis. A dual testicular andepididymal origin has been described for other proteins interacting withthe spermatozoa during epididymal maturation. Whether or not, thetesticular and the epididymal P26h are identical or exist in differentisoforms, as described for clusterin, remains to be determined.

The P26h being transcribed principally in testicular tissues (FIG. 2), atesticular cDNA was screened to clone the P26h cDNA. The longesttranscript obtained from the library was sequenced and revealed a cDNAof 1081 bp coding for a 244 amino acids protein. The predicted MW of thetranslational product is in agreement with the electrophoretic behaviourof P26h extracted from cauda epididymal spermatozoa. The P26h cDNA showshigh sequence homology with Adipsin, as expected from N-terminal aminoacid sequences and with a carbonyl reductase known to be expressed inpig lung (FIG. 4). The sequence homology between P26h and these twoproteins is 86% and 85% respectively. The deduced amino acid sequencealso shows a high homology of 87% with the Adipsin and 80% with theCarbonyl Reductase. Considering that P26h is a sperm protein involved ingamete interactions, the biological function of these two proteins waspuzzling. Adipsin has been described as a differentiation factor ofadipoblast in adipocytes and its expression has been shown to beinhibited by activators of protein kinase C. The carbonyl reductase is ahomotetramer that catalyzes the oxidation of secondary alcohols andaldehydes. This enzyme has been shown to be expressed specifically inthe lung and mainly distributed in the mitochondria. Notwithstanding thehigh level of homology with Adipsin and a carbonyl reductase, P26h showsa complete different tissues distribution. P26h protein and its encodingMRNA are not expressed in the lung nor in the adipocyte (FIGS. 2, andBerube, B., Sullivan, R., 1994, Biol. Reprod., 51: 1255-1263). Adipsinand carbonyl reductase are known to be members of the short-chaindehydrogenase/reductase (SDR) superfamily and P26h shows some of theseproperties.

The Short-Chain Dehydrogenase/Reductase superfamily (SDR) is formed by avariety of different proteins that exhibit residue identities of only15-30%. This low level of sequence identity between the membersindicates an early divergence. This is reflected by the wide range offunctions fulfilled by the members of this superfamily. There are threeclasses of enzymes covering a wide range of EC numbers: 1, 4.2, 5.1, and5.3, as well as members with unknown functions. Two consensus sequencesare conserved in this family, the NAD(H) or NADP(H) binding domain, aN-terminal segment GlyXXXGLYXXGly, and the catalytic domain, a sequenceTyrXXXLys. The P26h deduced amino acid sequence possesses theseconsensus domains as well as the Gly 129, Ser 136 and Pro 179 which areconserved in more than 90% of the SDR family members (FIG. 4).

Polyclonal antibodies have been produced against P26h and used todocument the function of this sperm protein during the fertilizationprocesses in the hamster. When added to an in vitro fertilizationmedium, the antibodies anti-P26h inhibits in a dose-dependent manner,the sperm-zona pellucida interaction. Furthermore, active immunizationof male hamsters against the purified P26h results in an immune responseassociated with reversible infertility. Using the anti-P26h antiserum, ahuman counterpart of P26h has also been identified and was showed to beabsent from sperm of men presenting with idiopathic infertility. Inhumans, this protein is also acquired by the spermatozoa during theepididymal transit. Taken together, these results clearly demonstratethe involvement of this sperm protein in the processes leading tofertilization. The P26h preparation that shows an immunocontraceptiveproperties is the same than the one used to determine N-terminalsequence by Edman degradation (FIG. 1). Furthermore, the polyclonalantiserum that allowed us to document the function of P26h in theprocesses of fertilization also reacts with the translational productencoded by the sequenced cDNA (FIG. 6). This clearly demonstrates thatthis SDR member is involved in mammalian sperm-egg interaction.

The mammalian spermatozoon is a highly polarized cell characterized bywell defined membrane domains. Many sperm surface proteins have beenproposed to play a role during the cascade of events occurring when themale gamete reaches the oocyte. Different sperm proteins have beenproposed as candidates involved in zona pellucida binding. Some of themshow enzymatic activity such as proacrosin, a trypsin like protease, amannosidase, a galactosyltransferase, and P95: a hexokinase. Thecatalytic activity of these enzymes may not necessarily be involved inzona pellucida interaction, it is rather the substrate affinity thatmediates this interaction. The biological function played by theseproteins in gamete interactions is thus quite different from theirenzymatic activity defined by their catalytic activity in cellmetabolism. This discrepancy is reflected by their subcellularlocalization on the spermatozoon. To mediate zona pellucida recognitionthese enzymes must be localized at the sperm surface where they areclassically known to be intracellular. This is well illustrated by theextracellular oriented sperm membrane mannosidase andgalactosyltransferase, as well as by hexokinase which is at the surfaceof the mouse spermatozoa where it is known to be associated with themitochondrial membrane. Like these potential zona pellucida ligand, P26his localized at the hamster sperm surface, to the membrane domaincovering the acrosome.

P26h belongs to the SDR superfamily characterized by highly differentmembers with a low level of identity. This reflects distant duplicationsand early divergence. As a consequence, SDR family represents a greatdiversity in enzymatic activities and functions. An interesting exampleof an alternative function for an enzyme, is glyceraldehyde -3-phosphatedehydrogenase. This protein which is classically known as a glycolyticenzyme has been shown to act as a t-RNA binding protein with a functionin cytoplasmic trafficking. SDR divergence is favourable for arising theof new functions; involvement in gamete interactions may be one ofthese. Considering that P26h has been previously shown to be involved ingamete interaction and to possess immunocontraceptive properties,cloning of a homologous cDNA in human allowed the identification of ahuman sperm protein with immunocontraceptive properties (FIG. 8). Theblood-testis barrier is not present in the epididymis, allowing theneutralization of spermatozoa following immunization against an antigeninvolved in post-testicular maturation of the male gamete. The fact thatthe human sperm protein is specifically expressed in the epididymis(FIG. 7), strongly supports its potential as an immunocontraceptivetarget.

The present invention will be more readily understood by referring tothe following example which is given to illustrate the invention ratherthan to limit its scope.

EXAMPLE I Immiunocontraception vaccine

The human counterpart of the hamster cDNA P26h, encodes for anepididymal-specific protein that is important in sperm function. It willbe possible to target this protein by specific antibodies using animmunocontraceptive approach. Men will be immunized with a peptidecorresponding to the epididymal protein. This peptide will be chosenwith regards to its antigenic properties. An immune response againstthat specific peptide will occur and no side effect is expected sincethe selected peptide shows high specificity for a sperm-epididymalprotein. The antibodies will reach the spermatozoa within the excurrentduct (epididymis) since the blood-testis barrier is not present at thelevel of the epididymis. The antibodies will neutralize the fertilizingability of the spermatozoa as already shown with the hamster P26h andwill confer an immuncontraceptive protection.

The peptide will be coupled to a carrier that will modulate thehalf-life of the circulating peptide. This will allow the control on theperiod of contraceptive protection. The peptide-carrier will beemulsified in an adjuvant and administrated by usual immunization route.

In men under such an immunocontraceptive regimen, the circulating titerof anti-peptide antibodies will be an indication of the contraceptiveefficiency. Expected reversibility will be predicted by standardimmunological determination of the titer of antibodies specific to thespecific peptide.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth, and as follows in the scopeof the appended claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 5                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 1081 base                                                         (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Coding Se - #quence                                             (B) LOCATION: 124...853                                                       (D) OTHER INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - GTCCCTGGAG GTTGGCTGTA GGATTCAGGT GGCTTGCTCA GGCTGGGATC AA - #GGACACAG         60                                                                          - TGAGCAGATC AACCTTAACC TCAGCCCCTC CCCTCGCCAC AGGAGGACAC TG - #GTGTCAGC        120                                                                          - AGC ATG AAG CTG AAT TTC ACT GGT CTC AGG GC - #T CTG GTG ACC GGG GCA          168                                                                          #Arg Ala Leu Val Thr Gly AlaGly Leu                                           #  15                                                                         - GGG AGA GGG ATT GGG CGA GGC ACT GCG AAA GC - #C CTG CAT GCC TCA GGA          216                                                                          Gly Arg Gly Ile Gly Arg Gly Thr Ala Lys Al - #a Leu His Ala Ser Gly           #                30                                                           - GCC AAA GTG GTG GCC GTG TCA CTC ATC AAC GA - #A GAC CTG GTC AGC CTG          264                                                                          Ala Lys Val Val Ala Val Ser Leu Ile Asn Gl - #u Asp Leu Val Ser Leu           #            45                                                               - GCC AAA GAG TGT CCG GGC ATA GAG CCT GTG TG - #T GTG GAC CTG GGT GAC          312                                                                          Ala Lys Glu Cys Pro Gly Ile Glu Pro Val Cy - #s Val Asp Leu Gly Asp           #        60                                                                   - TGG GAG GCC ACA GAG AAG GCA CTG GGC CGT AT - #T GGC CCC GTG GAC CTG          360                                                                          Trp Glu Ala Thr Glu Lys Ala Leu Gly Arg Il - #e Gly Pro Val Asp Leu           #    75                                                                       - CTG GTG AAC AAT GCG GCG GTG GCG CTA GTG CA - #G CCT TTC ATA CAG TCT          408                                                                          Leu Val Asn Asn Ala Ala Val Ala Leu Val Gl - #n Pro Phe Ile Gln Ser           #95                                                                           - ACC AAG GAG GTC TTT GAC AGG TCC TTC AAT GT - #G AAT GTG CGC TCT GTG          456                                                                          Thr Lys Glu Val Phe Asp Arg Ser Phe Asn Va - #l Asn Val Arg Ser Val           #               110                                                           - CTG CAA GTG TCC CAG ATG GTA GCC AAG GGC AT - #G ATT AAC CGT GGA GTG          504                                                                          Leu Gln Val Ser Gln Met Val Ala Lys Gly Me - #t Ile Asn Arg Gly Val           #           125                                                               - GCA GGA TCC ATT GTC AAC ATC TCC AGC ATG GT - #G GCC TAT GTC ACC TTC          552                                                                          Ala Gly Ser Ile Val Asn Ile Ser Ser Met Va - #l Ala Tyr Val Thr Phe           #       140                                                                   - CCT GGT CTG GCC ACG TAC AGC TCC ACC AAG GG - #T GCT ATA ACC ATG CTG          600                                                                          Pro Gly Leu Ala Thr Tyr Ser Ser Thr Lys Gl - #y Ala Ile Thr Met Leu           #   155                                                                       - ACC AAA GCC ATG GCC ATG GAG CTG GGA CCA TA - #C AAG ATC CGG GTG AAC          648                                                                          Thr Lys Ala Met Ala Met Glu Leu Gly Pro Ty - #r Lys Ile Arg Val Asn           160                 1 - #65                 1 - #70                 1 -       #75                                                                           - TCT GTA AAC CCT ACC GTG GTG CTG ACT GAC AT - #G GGC AAG AAA GTC TCT          696                                                                          Ser Val Asn Pro Thr Val Val Leu Thr Asp Me - #t Gly Lys Lys Val Ser           #               190                                                           - GCA GAC CCG GAA TTT GCC AAG AAG CTC AAG GA - #G CGC CAC CCA CTG AGG          744                                                                          Ala Asp Pro Glu Phe Ala Lys Lys Leu Lys Gl - #u Arg His Pro Leu Arg           #           205                                                               - AAG TTC GCA GAG GTG GAG GAC GTG GTC AAC AG - #C ATC CTC TTC CTG CTC          792                                                                          Lys Phe Ala Glu Val Glu Asp Val Val Asn Se - #r Ile Leu Phe Leu Leu           #       220                                                                   - AGC GAC AGC AGC GCC TCT ACC AGC GGC TCT GG - #C ATC CTG GTG GAC GCT          840                                                                          Ser Asp Ser Ser Ala Ser Thr Ser Gly Ser Gl - #y Ile Leu Val Asp Ala           #   235                                                                       #GAGACTTCCC        893TAGACGGC CCAGGTGCAG GGGACTCCTG                          Gly Tyr Leu Ala Ser                                                           240                                                                           - TGGCCTCACC CTTACATCAA GACCCCGCCT TCAACCCAAC CCAATAATTT TG - #TTCGAATC        953                                                                          - CTGTAGAGCC CCACCCCACA CACATCCATC CCCAACTTTA GACTCCGGGA TC - #CCGCCATT       1013                                                                          - CCATACCAGC TATGCTGAGA TAATTTGATT AAATAAGTAT CCCAAACCAC AA - #AAAAAAAA       1073                                                                          #        1081                                                                 - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 244 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: protein                                             -      (v) FRAGMENT TYPE: internal                                            -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Met Lys Leu Asn Phe Thr Gly Leu Arg Ala Le - #u Val Thr Gly Ala Gly         #                15                                                           - Arg Gly Ile Gly Arg Gly Thr Ala Lys Ala Le - #u His Ala Ser Gly Ala         #            30                                                               - Lys Val Val Ala Val Ser Leu Ile Asn Glu As - #p Leu Val Ser Leu Ala         #        45                                                                   - Lys Glu Cys Pro Gly Ile Glu Pro Val Cys Va - #l Asp Leu Gly Asp Trp         #    60                                                                       - Glu Ala Thr Glu Lys Ala Leu Gly Arg Ile Gl - #y Pro Val Asp Leu Leu         #80                                                                           - Val Asn Asn Ala Ala Val Ala Leu Val Gln Pr - #o Phe Ile Gln Ser Thr         #                95                                                           - Lys Glu Val Phe Asp Arg Ser Phe Asn Val As - #n Val Arg Ser Val Leu         #           110                                                               - Gln Val Ser Gln Met Val Ala Lys Gly Met Il - #e Asn Arg Gly Val Ala         #       125                                                                   - Gly Ser Ile Val Asn Ile Ser Ser Met Val Al - #a Tyr Val Thr Phe Pro         #   140                                                                       - Gly Leu Ala Thr Tyr Ser Ser Thr Lys Gly Al - #a Ile Thr Met Leu Thr         145                 1 - #50                 1 - #55                 1 -       #60                                                                           - Lys Ala Met Ala Met Glu Leu Gly Pro Tyr Ly - #s Ile Arg Val Asn Ser         #               175                                                           - Val Asn Pro Thr Val Val Leu Thr Asp Met Gl - #y Lys Lys Val Ser Ala         #           190                                                               - Asp Pro Glu Phe Ala Lys Lys Leu Lys Glu Ar - #g His Pro Leu Arg Lys         #       205                                                                   - Phe Ala Glu Val Glu Asp Val Val Asn Ser Il - #e Leu Phe Leu Leu Ser         #   220                                                                       - Asp Ser Ser Ala Ser Thr Ser Gly Ser Gly Il - #e Leu Val Asp Ala Gly         225                 2 - #30                 2 - #35                 2 -       #40                                                                           - Tyr Leu Ala Ser                                                             - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 912 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                           -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - GACAAAAGCT GGAGCTCCAC CGCGGTGGCG GCCGCTCTAG AACTAGTGGA TC - #CCCCGGGC         60                                                                          - TGCAGGAATT CGGCACGAGC CGACATGGAG CTGTTCCTCG CGGGCCGCCG GG - #TGCTGGTC        120                                                                          - ACCGGGGCAG GCAAAGGTAT AGGGCGCGGC ACGGTCCAGG CGCTGCACGC GA - #CGGGCGCG        180                                                                          - CGGGTGGTGG CTGTGAGCCG GACTCAGGCG GATCTTGACA GCCTTGTCCG CG - #AGTGCCCG        240                                                                          - GGGATAGAAC CCGTGTGCGT GGACCTGGGT GACTGGGAGG CCACCGAGCG GG - #CGCTGGGC        300                                                                          - AGCGTGGGCC CCGTGGACCT GCTGGTGAAC AACGCCGCTG TCGCCCTGCT GC - #AGCCCTTC        360                                                                          - CTGGAGGTCA CCAAGGAGGC CTTTGACAGA TCCTTTGAGG TGAACCTGCG TG - #CGGTCATC        420                                                                          - CAGGTGTCGC AGATTGTGGC CAGGGGCTTA ATAGCCCGGG GAGTACCAGG GG - #CCATCGTG        480                                                                          - AATGTCTCCA GCCAGTGCTC CCAGCGGGCA GTAACTAACC ATAGCGTCTA CT - #GCTCCACC        540                                                                          - AAGGGTGCCC TGGACATGCT GACCAAGGTG ATGGCCCTAG AGCTCGGGCC CC - #ACAAGATC        600                                                                          - CGAGTGAATG CAGTAAACCC CACAGTGGTG ATGACGTCCA TGGCCAGCCC AC - #CTGGAGTG        660                                                                          - ACCCCCACAA GCCAAGACTA TGCTGAACCG AATCCCACTT GGCAAGTTTG CT - #GAGGTAGA        720                                                                          - GCACGTGGTG AACGCCATCC TCTTTCTGCT GAGTGACCGA AGTGGCATGA CC - #ACGGGTTC        780                                                                          - CACTTTGCCG GTGGAAGGGG GCTTCTGGGC CTGCTGAGCT CCCTCCACAC AC - #CTCAAGCC        840                                                                          - CCATGCCGTG CTCATCCTAC CCCCAATCCC TCCAATAAAC CTGATTCTGC TC - #CCAAAAAA        900                                                                          #      912                                                                    - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Glu Leu Phe Leu Ala Gly Arg Arg Val Cy - #s                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 14 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Cys Ser Gln Asp Tyr Ala Glu Pro Asn Pro Th - #r Trp Gln Val                 #                10                                                           __________________________________________________________________________

What is claimed is:
 1. A method of immunocontraception of a male orfemale subject, which comprises administering to said male or femalesubject an antigenic fragment of a P34 protein to elicit animmunocontraception response by said male or female subject.
 2. Themethod of claim 1, wherein said P34 protein is encoded by the sequenceas set forth in SEQ ID NO:3, and wherein said antigenic, fragment has anamino acid sequence selected from the group consisting of SEQ ID NO:4andSEQ ID NO:5.
 3. An immunocontraceptive vaccine for a male or femalesubject, which comprises an antigenic fragment of a P34 protein inassociation with a suitable pharmaceutically acceptable carrier, whereinsaid vaccine elicits an immunocontraception response by said male orfemale subject after its administration.